1. Technical Field
The subject invention relates to the identification of a gene involved in the elongation of long-chain polyunsaturated fatty acids (i.e., “elongase”) and to uses thereof. In particular, the elongase enzyme is utilized in the conversion of one fatty acid to another. For example, elongase catalyzes the conversion of γ-linolenic acid (GLA, 18:3n-6) to dihomo-γ-linolenic acid (DGLA, 20:3n-6) and the conversion of stearidonic acid (STA, 18:4n-3) to eicosatetraenoic acid (ETA, 20:4n-3). Elongase also catalyzes the conversion of arachidonic acid (AA, 20:4n-6) to adrenic acid (ADA, 22:4n-6), the conversion of eicosapentaenoic acid (EPA, 20:5n-3) to ω3-docosapentaenoic acid (22:5n-3), the conversion of linoleic acid (LA, 8:2n-6) to eicosadienoic acid (EDA, 20:2n6), and the conversion of α-linolenic acid (ALA, 18:3n-3) to eicosatrienoic acid (ETrA, 20:3n-3). ALA, for example, may be utilized in the production of other polyunsaturated fatty acids (PUFAs), such as ETrA. ETrA may then be converted to ETA by a Δ8-desaturase. ETA may then be utilized in the production of other polyunsaturated fatty acids, such as EPA, which may be added to pharmaceutical compositions, nutritional compositions, animal feeds, as well as other products such as cosmetics.
2. Background Information
The elongases which have been identified in the past differ in terms of the substrates upon which they act. Furthermore, they are present in both animals and plants. Those found in mammals have the ability to act on saturated, monounsaturated and polyunsaturated fatty acids. In contrast, those found in plants are specific for saturated or monounsaturated fatty acids. Thus, in order to generate polyunsaturated fatty acids in plants, there is a need for a PUFA-specific elongase.
In both plants and animals, the elongation process is believed to be the result of a four-step mechanism (Lassner et al., The Plant Cell 8:281-292 (1996)). CoA is the acyl carrier. Step one involves condensation of malonyl-CoA with a long-chain acyl-CoA to yield carbon dioxide and a β-ketoacyl-CoA in which the acyl moiety has been elongated by two carbon atoms. Subsequent reactions include reduction to β-hydroxyacyl-CoA, dehydration to an enoyl-CoA, and a second reduction to yield the elongated acyl-CoA. The initial condensation reaction is not only the substrate-specific step but also the rate-limiting step.
As noted previously, elongases, more specifically, those which utilize PUFAs as substrates, are critical in the production of long-chain polyunsaturated fatty acids which have many important functions. For example, PUFAs are important components of the plasma membrane of a cell where they are found in the form of phospholipids. They also serve as precursors to mammalian prostacyclins, eicosanoids, leukotrienes and prostaglandins. Additionally, PUFAs are necessary for the proper development of the developing infant brain as well as for tissue formation and repair. In view of the biological significance of PUFAs, attempts are being made to produce them, as well as intermediates leading to their production, efficiently.
A number of enzymes are involved in PUFA biosynthesis including elongases (ELO) (FIG. 1). For example, linoleic acid (LA, 18:2n-6) is produced from oleic acid (OA, 18:1n-9) by a Δ12-desaturase. Eicosadienoic acid (EDA, 20:2n-6) is produced from linoleic acid (LA, 18:2n-6) by a Δ9-elongase. Dihomo-γ-linolenic acid (DGLA, 20:3n-6) is produced from eicosadienoic acid (EDA, 20:2n-6) by a Δ8-desaturase. Arachidonic acid (AA, 20:4n-6) is produced from dihomo-γ-linolenic acid (DGLA, 20:3n-6) by a Δ5-desaturase.
It must be noted that animals cannot desaturate beyond the Δ9 position and therefore cannot convert oleic acid (OA, 18:1n-9) into linoleic acid (LA, 18:2n-6). Likewise, α-linolenic acid (ALA, 18:3n-3) cannot be synthesized by mammals, since they lack Δ15-desaturase activity. However, α-linolenic acid can be converted to stearidonic acid (STA, 18:4n-3) by a Δ6-desaturase (see PCT publication WO 96/13591; see also U.S. Pat. No. 5,552,306), followed by elongation to eicosatetraenoic acid (ETA, 20:4n-3) in mammals and algae. This polyunsaturated fatty acid (i.e., ETA, 20:4n-3) can then be converted to eicosapentaenoic acid (EPA, 20:5-3) by a Δ5-desaturase. Other eukaryotes, including fungi and plants, have enzymes which desaturate at carbons 12 (see PCT publication WO 94/11516 and U.S. Pat. No. 5,443,974) and 15 (see PCT publication WO 93/11245). The major polyunsaturated fatty acids of animals therefore are either derived from diet and/or from desaturation and elongation of linoleic acid or α-linolenic acid. In view of the inability of mammals to produce these essential long-chain fatty acids, it is of significant interest to isolate genes involved in PUFA biosynthesis from species that naturally produce these fatty acids and to express these genes in a microbial, plant or animal system which can be altered to provide production of commercial quantities of one or more PUFAs. Consequently, there is a definite need for elongase enzymes, the genes encoding the enzymes, as well as recombinant methods of producing the enzymes.
In view of the above discussion, a definite need exists for oils containing levels of PUFAs beyond those naturally present as well as those enriched in novel PUFAs. Such oils can only be made by isolation and expression of elongase genes.
One of the most important long-chain PUFAs is eicosapentaenoic acid (EPA). EPA is found in fungi and also in marine oils. Docosahexaenoic acid (DHA) is another important long-chain PUFA. DHA is most often found in fish oil and can also be purified from mammalian brain tissue. Arachidonic acid (AA) is a third important long-chain PUFA. AA is found in filamentous fungi and can also be purified from mammalian tissues including the liver and the adrenal glands.
AA, EPA and/or DHA, for example, can be produced via either the alternate delta 8 pathway or the conventional delta 6 pathway (FIG. 1). Elongase, which are active on substrate fatty acids in the conventional delta 6 pathway for the production of long-chain PUFAs, particularly AA, EPA and DHA, have previously been identified. The conventional delta 6 pathway for converting LA to DGLA and ALA to ETA utilizes the Δ6-desaturase enzyme to convert LA to GLA, and ALA to STA, and the Δ6-elongase enzyme to convert GLA to DGLA, and STA to ETA. However, in certain instances, the alternate delta 8 pathway may be preferred over the conventional delta 6 pathway. For example, if particular residual omega-6 or omega-3 fatty acid intermediates, such as GLA or STA, are not desired during production of DGLA, ETA, AA, EPA, ω3-docosapentaenoic acid, ω6-docosapentaenoic acid, ADA and/or DHA, the alternate delta 8 pathway may be used as an alternative to the conventional delta 6 pathway, to bypass GLA and STA formation.
In the present invention, a new source of Δ9-elongase has been identified for the production of long-chain PUFAs, in particular DGLA, ETA, AA, EPA, ω3-docosapentaenoic acid, ω6-docosapentaenoic acid, ADA and/or DHA. Such oils can be made, in part, by isolation and expression of the Δ9-elongase gene. The Δ9-elongase enzyme of the present invention converts, for example, LA to EDA. The production of DGLA from EDA, and AA from DGLA, is then catalyzed by a Δ8-desaturase and a Δ5-desaturase, respectively.
The search for a long-chain PUFA-specific Δ9-elongase in Thraustochytrid sp. began based upon a review of the homologies shared between this gene and by expression screening for PUFA-elongase activity.
All patents and publications referred to herein are hereby incorporated in their entirety by reference.